Knowledge gathered about the voltage-gated K+ channel Kv1.3 in recent years indicates the essential role of this channel in T cell activation and the killing process of cytotoxic cells. It is therefore of utmost importance to understand the details of the physiological role of Kv1.3 in T cell signaling and the mutual interplay between the channel and other signaling molecules. The prospect of using Kv1.3 as a target for immunosuppressive drugs in therapy further emphasizes the importance of studies focusing on this channel. As channel blockers of high affinity and selectivity are valuable tools for studying ion channel function and, in addition, they may serve as starting points in the development of new immunosuppressive agents, the search for such compounds is always justified. We identified and characterized a new peptide blocker of the Kv1.3 channel and proved that anuroctoxin selectively blocks Kv1.3 with high affinity (Kd = 0.73 nM). Regarding T cell physiology, the most important factor in the pharmacological profile of anuroctoxin is its selectivity for Kv1.3 over IKCa1, the Ca2+-activated K+ channel of human T lymphocytes. Mechanism of Kv1.3 block by anuroctoxin is similar to the block of other scorpion toxins, anuroctoxin is classical pore blocker. Because of its high selectivity for Kv1.3 (anuroctoxin did not block either Kv1.x or Kv2.1 channels), anuroctoxin might be used in clinical practice as an effective immunosuppressive agent to block lymphocyte activation and proliferation in the future. It is generally accepted that functional potassium channels and control of the membrane potential are essential to mitogenic induction of lymphocytes via the regulation of cell volume and cell-mediated cytotoxicity. In our experiments we examined the spatial distribution and association of Kv1.3 and molecules essential in T cell signal transduction to draw conclusions about the role of Kv1.3 in T cell activation based on these molecular interactions. We demonstrated in Jurkat cells and CTLs that the Kv1.3 channel is raft associated and distribution of the channel is nonrandom. We demonstrated a significant domain-level colocalization of Kv1.3/FLAG and CD3 in the Jurkat cell membrane. We showed the enrichment of Kv1.3/FLAG in the immunological synapse formed by CTL-JY cells. We proved that the Kv1.3 channels enter the IS during its development and they undergo a dynamical rearrangement. In conclusion, the results of our studies gave great contribution to the understanding of Kv1.3 function and regulation by the identification of a new high affinity and specificity blocker and the characterization of the distribution of these channels in the plasma membrane of lymphocytes.